Application of fluorescence sensor based on neomycin DNA aptamer

Abstract

The invention discloses application of a fluorescence sensor based on a neomycin DNA aptamer, and belongs to the technical field of aptamer sensors. The neomycin DNA aptamer with excellent performance is combined with the nano material with unique chemical performance, the prepared aptamer fluorescence sensor has high sensitivity and specificity, a satisfactory linear relation is shown between the fluorescence value of the system and the neomycin concentration in the range of 0.005-200ng / mL, the detection analysis operation is simple, the detection time is short, and the application prospect is wide. The method can be used for detecting neomycin in food and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the application of a fluorescence sensor based on neomycin DNA aptamer, belonging to the technical field of aptamer sensors. Background Art

[0002] Neomycin is an aminoglycoside antibiotic, which is relatively common in dairy farming due to its low cost, high efficiency, and broad spectrum. Neomycin is strictly restricted in human clinical use due to its severe ototoxicity and nephrotoxicity. However, in the process of livestock breeding, it is wantonly used due to its good therapeutic effect and low price. After consuming animal-derived foods contaminated with veterinary drugs, it accumulates in the human body and causes diseases, and in severe cases, it can lead to death. In order to monitor the residue of neomycin in edible tissues of animals, it is extremely necessary to establish a method for detecting the residue of neomycin in edible tissues of animals. Currently, the conventional methods for determining neomycin are mainly ELISA immunoassay and instrumental analysis methods, but they have the disadvantages of cumbersome operation, expensive instruments, and high technical requirements for operators.

[0003] Nucleic acid aptamers are artificially synthesized short-chain nucleic acids, which are short oligonucleotides screened from a wide range of nucleic acid libraries using the systematic evolution of ligands by exponential enrichment (SELEX) technology. As molecular recognition elements, aptamers can bind to various target substances with high specificity and high affinity, and are divided into two types: single-stranded DNA and RNA. Among them, RNA is easily degraded by nucleases and has poor stability, which limits its application in aptamer sensors. Single-stranded DNA aptamers have more application potential than RNA aptamers due to their low synthesis cost, high stability, and easy chemical modification, and are widely used in popular fields such as analytical chemistry, biomedicine, and food safety detection. It is worth noting that all reported neomycin aptamers are RNA aptamers, and there is no research on neomycin DNA aptamers. Since the cycle of de novo SELEX screening is long and the cost is high, it would be very meaningful if the classical neomycin RNA aptamer could be transformed into a DNA aptamer with excellent performance. Summary of the Invention

[0004] To solve the above technical problems, the object of the present invention is to provide a method for modifying neomycin aptamer and an application study of a DNA aptamer fluorescence sensor.

[0005] The present invention provides a DNA aptamer for detecting neomycin, and its nucleotide sequence is 5'-GGACTGGGCGAGAAGTTTAGTCC-3' (SEQ ID NO.1).

[0006] The present invention also provides a fluorescence biosensor based on neomycin aptamer, including:

[0007] (a) A T1-T2 double-stranded recognition solution prepared from the neomycin aptamer shown in SEQ ID NO.1 and the complementary strand shown in SEQ ID NO.2;

[0008] (b) A magnetic microsphere capture probe MBs-H1 with surface-modified H1 hairpin DNA; the H1 hairpin DNA has the nucleotide sequence shown in SEQ ID NO.3;

[0009] (c) A metal-organic framework fluorescent signal probe MOF@CuNCs-H2 with surface-loaded copper nanoclusters and modified H2 hairpin DNA; the H2 hairpin DNA has the nucleotide sequence shown in SEQ ID NO.4.

[0010] In one embodiment, the preparation method of the metal-organic framework-copper nanoclusters MOF@CuNCs is as follows:

[0011] ① Mix the Cu(NO3)2 solution with the histidine solution, then add the ascorbic acid solution, and incubate with shaking at 37 °C for 6 h to obtain light brown CuNCs;

[0012] ② Mix the CuNCs prepared in step ① with the Zn(NO3)2-methanol solution and stir, then add the 2-methylimidazole-methanol solution, stir at room temperature for 2 h and then let stand overnight, centrifuge to collect the light brown precipitate, wash 3 times with methanol, and dry in vacuum to obtain the powder product MOF@CuNCs.

[0013] In one embodiment, the fluorescent signal probe MOF@CuNCs-H2 is prepared by dissolving the MOF@NCs in the EDC and NHS solutions, incubating with shaking to activate the surface carboxyl groups; adding the activated MOF@NCs to the carboxylated H2 hairpin solution and coupling with shaking overnight, followed by washing and centrifugation.

[0014] The present invention also provides a method for detecting neomycin based on DNA aptamers and MOF@CuNCs, comprising the following steps:

[0015] Mix the sample to be tested with the pre-prepared T1-T2 complex for 30 min, then co-incubate with MBs-H1 and MOF@CuNCs-H2, add an appropriate buffer, continuously incubate with shaking at 37 °C for 2 h, then perform magnetic separation 3 times, and collect the obtained supernatant for fluorescence intensity analysis.

[0016] In one embodiment, the volume ratio of MBs-H1 to MOF@CuNCs-H2 is 1:1.5 to 1:2.

[0017] In one embodiment, the fluorescence intensity is detected at an excitation wavelength of 400 nm and an emission wavelength of 495 nm.

[0018] In one embodiment, the neomycin concentration is calculated according to the standard curve y = 465.73lgC + 2937.63.

[0019] The present invention also provides an application of the biosensor in detecting neomycin residues in foods.

[0020] In one embodiment, the foods include but are not limited to animal foods or dairy products.

[0021] In one embodiment, the animal foods include but are not limited to livestock and poultry meat and by-products, and aquatic products.

[0022] In one embodiment, the dairy products include but are not limited to milk, milk powder, and pasteurized milk.

[0023] In one embodiment, the food is milk or honey.

[0024] Beneficial effects:

[0025] (1) Due to the limitations of insufficient stability and high synthesis cost of RNA aptamers in the prior art, it is not conducive to achieving stable detection in complex detection matrices, which greatly limits the application of aptamers in biosensors. The present invention successfully converts neomycin RNA aptamer into DNA aptamer for the first time, and analyzes the affinity and spatial structure characteristics of the obtained aptamer. The present invention provides a new idea for the modification of aptamers, and has great research and promotion value.

[0026] (2) The present invention synthesizes MOF@CuNCs with significantly improved fluorescence performance, and the fluorescence quantum yield and fluorescence lifetime are significantly improved compared with CuNCs. The prepared fluorescent probe MOF@CuNCs-H2 is combined with catalytic hairpin assembly (CHA) amplification technology to achieve highly sensitive and rapid detection of neomycin. The CHA reaction process does not require the participation of polymerase, and the trigger single-stranded DNA binds to the base region exposed by the shorter strand in the hairpin structure to achieve the displacement process. The created detection method does not require the participation of protease and the reaction conditions are mild, which greatly improves the output efficiency of fluorescence signals.

[0027] The present invention combines a neomycin DNA aptamer with excellent performance and a nanomaterial with unique chemical properties. The prepared aptamer fluorescence sensor has high sensitivity and specificity, and the detection and analysis operation is simple, the detection time is short, and it can be used for the detection of neomycin in milk and honey, and has broad application prospects. Description of the Drawings

[0028] Figure 1For aptamer effect characterization; (A) and (B) are the affinity curves of the DNA aptamer of neomycin after modification, (C) is the CD spectral curve before and after the aptamer recognizes neomycin, and (D) is the CD temperature-variable curve before and after the aptamer recognizes neomycin.

[0029] Figure 2 It is the schematic diagram of fluorescence signal amplification detection constructed based on the modified DNA aptamer.

[0030] Figure 3 For the characterization of MOF@CuNCs material; among them, (A) is the SEM image of ZIF-8; (B), (C), and (D) are the SEM images of MOF@CuNCs; (E) is the EDS mapping of C, N, Zn, and Cu elements; (F) is the infrared spectra of MOF@CuNCs and ZIF-8; (G) is the XRD patterns of MOF@CuNCs and ZIF-8; (H) is the Zeta potential data of CuNCs, MOF@CuNCs, and ZIF-8.

[0031] Figure 4 It is the fluorescence intensity under different reaction conditions; among them, (A) is the volume ratio of the capture probe H1-MB to the signal probe H2-MOF@CuNCs, and (B) is the optimization of the CHA reaction time.

[0032] Figure 5 For the performance analysis of the detection method; (A) is the relationship between the fluorescence intensity and the neomycin concentration in the range of 0 - 500 ng / mL, (B) is the standard curve of neomycin, (C) is the fluorescence intensity spectrogram, (D) is the specificity of the aptamer sensor, (E) is the detection stability for 30 days, and (F) is the detection reproducibility. Detailed implementation method

[0033] Example 1 Modification of neomycin aptamer and performance characterization of DNA aptamer

[0034] The original neomycin RNA aptamer sequence is 5’-GGACUGGGCGAGAAGUUUAGUCC-3’. After replacing the U base with the T base, the modified DNA aptamer sequence is: 5’-GGACTGGGCGAGAAGTTTAGTCC-3’, which is prepared according to the following steps.

[0035] (1) Isothermal Titration Calorimetry (ITC): Both neomycin in the titration syringe and the aptamer solution in the sample cell were dissolved in the aptamer binding buffer (containing 20 mM Tris-HCl, 50 mM MgCl2, 5 mM KCl, 2 mM CaCl2, pH 7.6). Before analysis, the samples were centrifuged at 10000 r / min for 5 min to remove air bubbles. In the formal experiment, 200 μM neomycin in the syringe was titrated into 10 μM aptamer in the sample cell. PBS was used to replace the aptamer in the above steps and set as the control group to exclude the interference of dilution heat.

[0036] It can be seen from Figure 1 (A)(B) that the entire aptamer-neomycin binding process is exothermic, and the affinity of the obtained DNA aptamer of neomycin after modification is 6.06 ± 1.1 μM.

[0037] (2) Circular Dichroism (CD): The concentration of the aptamer solution was 10 μM, and the concentration of the neomycin solution was 100 μM. The experiment was carried out at room temperature. After the instrument was turned on, it was purged with high-purity nitrogen (99.99%) for 20 min, and then the xenon lamp was lit. The specific parameter settings of the Chirascan software were as follows: the bandwidth was 1.0 nm, and the measurement range of the spectrum was 220 - 320 nm. Before starting to measure the sample, the instrument background was measured first. The sample chamber was left empty, and the signal of air (nitrogen) was directly collected. Then, the CD signals of the aptamer, neomycin, and the aptamer-neomycin complex were measured under the same conditions, and the CD signal of the BB binding buffer was used as the background signal.

[0038] It can be seen from Figure 1 (C) that the binding of neomycin to the aptamer can induce changes in the structure of the aptamer, enhance the double-helix stacking effect of the aptamer, and make the stem region of the secondary structure of the aptamer more compact and stable. From the CD T m curve, it can be seen that the CD T m value after the binding of the aptamer and neomycin increased by 4.8 °C compared with that of the single aptamer, which may be due to the non-covalent bond interactions between the aptamer-neomycin, such as hydrogen bonds and van der Waals forces, enhancing the thermal stability of the aptamer structure.

[0039] Example 2 Synthesis and Characterization of Fluorescence-Enhanced Nanomaterials

[0040] Synthesis of MOF@CuNCs: Fluorescent CuNCs were prepared using L-histidine as a stabilizer and reducing agent. Cu(NO3)2 solution (4.5 μL, 0.10 mol / L) was added to the histidine solution (1.0 mL, 0.10 mol / L), and the mixture was shaken at room temperature for 30 min. Then, ascorbic acid solution (50.0 μL, 0.10 mol / L) was added. After incubation with shaking at 37 °C for 6 h, the obtained light brown CuNCs were ultrafiltered and concentrated and stored at 4 °C.

[0041] Take 0.1 mL of CuNCs and mix it with 1 mL of Zn(NO3)2-methanol solution (30 mM) and stir for 5 min. Then, add 1 mL of 2-methylimidazole-methanol solution (90 mM), stir at room temperature for 2 h, and then let it stand and react overnight. The reaction solution was centrifuged (7000 rpm, 10 min) to obtain a light brown precipitate, which was washed 3 times with methanol and dried under vacuum at 30 °C to obtain the powder product MOF@CuNCs.

[0042] Characterize the prepared composite material MOF@CuNCs.

[0043] (1) Scanning electron microscope results showed that ZIF-8 synthesized from Zn(NO3)2 and 2-methylimidazole was a rhombic dodecahedron with an average size of 486 nm ( Figure 3 A). Compared with ZIF-8, the surface of the obtained MOF@CuNCs after introducing CuNCs was rougher, and the particle size increased with the extension of the reaction time, with an average size of 574 - 960 nm ( Figure 3 B-3D).

[0044] (2) The EDS spectrum showed that Cu, Zn, C, and N elements were evenly distributed in the composite material MOF@CuNCs ( Figure 3 E).

[0045] (3) Since MOF@CuNCs was synthesized by the coordination of Zn 2+ with the carboxyl group on the histidine ligand of CuNCs and the N atom of 2-methylimidazole for self-assembly. In the infrared spectrum of MOF@CuNCs, a characteristic peak of Zn-O appeared at 524 cm -1 , indicating that the coordination between the carboxyl group in histidine and Zn(II) in ZIF-8 was the main reason for the successful self-assembly of MOF@CuNCs. In addition, the broad peak located at 2900 - 3750 cm -1 was the characteristic peak of the amino group of histidine, further proving the successful encapsulation of CuNCs in ZIF-8.

[0046] (4) The crystal structure of the synthesized MOF@CuNCs dodecahedra was further explored using Powder X-ray diffraction (PXRD). As Figure 3 shown in G, the characteristic peaks of the nanocomposite are consistent with those of pure ZIF-8, indicating that the incorporation of CuNCs does not affect the crystal growth of ZIF-8.

[0047] (5) Zeta potential data showed that with the introduction of CuNCs, the potential of ZIF-8 decreased from 31.33 mV to -9.56 mV, indicating an electrostatic interaction between CuNCs and ZIF-8. In summary, it was shown that CuNCs were successfully encapsulated within the framework material ZIF-8.

[0048] Example 3 Construction of Fluorescent Sensor

[0049] Before use, both hairpin nucleic acid strands H1 and H2 were dissolved in Tris-HCl buffer, denatured at 95 °C for 10 min, and then cooled to room temperature to ensure that they could fold into stable DNA hairpin structures. Avidin-coated MBs were linked to biotinylated H1, denoted as MBs-H1. The modification of hairpin H2 on MOF@NCs was as follows: 10 mg of dry MOF@NCs was dissolved in 100 μL of 50 mM EDC and NHS (1:1) solution and incubated with shaking for 2 h. The activated MOF@NCs were added to the carboxylated H2 hairpin solution and coupled with shaking overnight. After washing and centrifugation, MOF@NCs-H2 was obtained.

[0050] 200 nM of neomycin aptamer (T2, shown in SEQ ID NO.1) and its complementary strand (T1, shown in SEQ ID NO.2) were uniformly mixed in equimolar amounts. Subsequently, the mixture was denatured at 95 °C for 5 min and slowly cooled to room temperature (20 - 25 °C) and incubated for 2 h until they were fully complementary to form the T1-T2 recognition solution. Then, different concentrations of neomycin (50 μL) were uniformly mixed with 150 μL of MBs-H1 and 300 μL of MOF@CuNCs-H2 and reacted at 20 °C - 25 °C for at least 90 min. After the reaction, the reaction product was subjected to magnetic separation three times. 200 μL of the supernatant solution after the reaction was taken, and the change value of the fluorescence emission peak intensity at 495 nm under the excitation wavelength of 400 nm was measured on an F-7000 instrument.

[0051] Example 4 Optimization of Fluorescent Sensor Detection Conditions

[0052] (1) Optimization of the volume ratio of the capture probe MB-H1 to the fluorescent signal probe MOF@CuNCs-H2.

[0053] The total volume of the CHA reaction system was 500 μL. The volume of MB-H1 was fixed at 200 μL. The concentrations of MB-H1 and MOF@CuNCs-H2 were kept constant. Then, the volume ratio gradients of MB-H1:MOF@CuNCs-H2 were set as 1:0.5, 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, and 1:2.5, respectively. The results showed that when the volume ratio was between 1:1.5 and 1:2.5, the fluorescence intensity reached over 4000. When the volume ratio exceeded 1:1.75, the fluorescence change value tended to be stable and decreased slightly. This was mainly because the excessive MOF@CuNCs-H2 increased the fluorescence background signal of the blank group. The optimal concentration ratio was selected as 1:1.75( Figure 4 A).

[0054] (2) Optimization of reaction time

[0055] The reactions were carried out for 30 min, 60 min, 90 min, 120 min, and 150 min, respectively. The results showed that when the reaction time was over 90 min, the fluorescence intensity reached over 4000. If the time was too short, the aptamer and the target could not be recognized sufficiently, and the complementarity between H1 and H2 was also insufficient. Finally, 1.5 h was selected as the optimal reaction time for the CHA reaction( Figure 4 B).

[0056] Example 5 Establishment of the standard curve of neomycin

[0057] Under the conditions that the volume ratio of the capture probe MB-H1 to the signal probe MOF@CuNCs-H2 reached 1:1.75 and the reaction time was 1.5 h, as the concentration of neomycin increased, the fluorescence intensity of the detection system at 495 nm gradually increased, indicating that the aptasensor established by this method had a good response to the concentration of neomycin.

[0058] As Figure 5 shown in 2 B, in the range of 0.005 - 200 ng / mL, there was a satisfactory linear relationship between the fluorescence value of the system and the concentration of neomycin (y = 465.73lgC + 2937.63, R 2 = 0.995), and the LOD was 0.912 pg / mL (LOD = 3*σ / S, where σ was the standard deviation of the blank sample signal and S was the slope of the calibration curve). The RSD of 11 repeated determinations of 0.05 ng / mL neomycin was 2.77%.

[0059] Example 6 Verification of the specificity, stability, and reproducibility of the fluorescence sensor

[0060] To determine the specificity of the response of the constructed neomycin aptamer fluorescence sensing platform, other possible antibiotic interferents, such as chloramphenicol (CHL), doxycycline (DOX), oxytetracycline (OTC), enrofloxacin (ENR), azithromycin (AZT), and roxithromycin (RXM), were tested and analyzed under the same experimental conditions. The fluorescence response value of 0.1 ng / mL neomycin was significantly higher than that of the other six antibiotics (1 ng / mL), and when a mixed solution of neomycin and the above six antibiotics was present, the obtained fluorescence signal was significantly higher than that of the other antibiotics, indicating that the developed sensor has the specificity to meet the analytical detection requirements ( Figure 5 C).

[0061] A fluorescence sensor was prepared according to the method of Example 3 and used for the detection of spiked neomycin samples to evaluate the stability and reproducibility of the sensor. The aptamer sensors prepared in the same batch were stored at 4 °C, and the response signal to 0.1 ng / mL neomycin was recorded every 5 days to explore the stability of the aptamer sensor ( Figure 5 D). As the storage time prolonged, the fluorescence signal intensity showed a slight downward trend. After 30 days of storage, 98.7% of the initial fluorescence signal could still be maintained, indicating that the established aptamer sensor had satisfactory storage stability. For the reproducibility of the aptamer sensor, the measurement results of the prepared sensors were basically consistent, and the relative standard deviation (RSD) was 2.34%. The results are shown in Figure 5 E.

[0062] Example 7 Application of the Fluorescence Sensor in the Detection of Actual Samples

[0063] To verify the practical application of the method for detecting neomycin by the fluorescence sensor based on neomycin DNA aptamer of the present invention, different concentrations of neomycin (0.1, 0.5, 1, 10 ng / mL) were added to milk and honey. The above actual samples were respectively measured for neomycin in the samples by using the fluorescence aptasensor based on MOF@CuNCs prepared above and enzyme-linked immunosorbent assay (ELISA). The detection conditions of the fluorescence aptasensor based on MOF@CuNCs were as follows: The neomycin aptamer and its complementary strand were evenly mixed in equimolar amounts, denatured at 95 °C for 5 min and then cooled to room temperature, and incubated for 2 h. Then, 50 μL of 0.1, 0.5, 1, 10 ng / mL of neomycin were respectively and evenly mixed with 150 μL of MBs-H1 and 300 μL of MOF@CuNCs-H2, and oscillated and reacted at 37 °C for 2 h. After the reaction was completed, the reaction product was subjected to magnetic separation 3 times. 200 μL of the supernatant solution after the reaction was taken, and the change value of the fluorescence emission peak intensity at 495 nm under the excitation wavelength of 400 nm was measured on an F-7000 instrument. Finally, the fluorescence change value was substituted into the established neomycin standard curve, and the concentration under the corresponding fluorescence value condition was calculated. The enzyme-linked immunosorbent assay was performed using a neomycin kit (Shanghai Enzyme-linked Biology, product number ml514007).

[0064] The measurement results are shown in Table 1.

[0065] Table 1 Detection effects of different methods

[0066]

[0067] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A DNA aptamer for detecting neomycin, characterized in that, The nucleotide sequence is as shown in SEQ ID NO.

1.

2. A neomycin-based aptamer fluorescence biosensor, characterized in that, It includes: (a) A T1-T2 double-stranded recognition solution prepared from the neomycin aptamer shown in SEQ ID NO.1 and the complementary strand shown in SEQ ID NO.2; (b) A magnetic microsphere capture probe MBs-H1 with surface-modified H1 hairpin DNA; the H1 hairpin DNA has the nucleotide sequence shown in SEQ ID NO.3; (c) A metal-organic framework fluorescence signal probe MOF@CuNCs-H2 with surface-loaded copper nanoclusters and modified H2 hairpin DNA; the H2 hairpin DNA has the nucleotide sequence shown in SEQ ID NO.

4.

3. The biosensor according to claim 2, wherein The preparation method of the metal-organic framework - copper nanoclusters MOF@CuNCs is as follows: ① Mix the Cu(NO3)2 solution with the histidine solution, then add the ascorbic acid solution, and incubate with shaking at 35 - 40 °C for 5 - 7 h; ② Mix the CuNCs prepared in step ① with the Zn(NO3)2 - methanol solution, add the 2-methylimidazole - methanol solution, stir at 20 - 25 °C and then let it stand, and collect the precipitate by centrifugation.

4. According to the method described in claim 3, wash the precipitate prepared in step ② with methanol and then dry it to obtain the powder product MOF@CuNCs.

5. The biosensor according to claim 2, characterized in that, The fluorescence signal probe MOF@CuNCs-H2 is obtained by dissolving the MOF@NCs in the EDC and NHS solutions, incubating with shaking, and then adding it to the carboxylated H2 hairpin solution for shaking coupling, followed by washing and centrifugation; the nucleotide sequence of the H2 hairpin is as shown in SEQ ID NO.

4.

6. A neomycin detection method based on DNA aptamer and MOF@CuNCs, characterized in that, React the aptamer fluorescence biosensor described in claim 2 with the sample to be tested and detect the fluorescence intensity.

7. The method according to claim 6, wherein It includes the following steps: Mix the sample to be tested with the T1-T2 complex, then co-incubate it with the capture probe MBs-H1 and the fluorescence signal probe MOF@CuNCs-H2, add an appropriate buffer solution, incubate with shaking at 35 - 40 °C for 1 - 3 h, then perform magnetic separation, and collect the supernatant for fluorescence intensity analysis.

8. The method according to claim 7, wherein The volume ratio of MBs-H1 to MOF@CuNCs-H2 is 1:1.5 - 1:

2.

9. The method according to any one of claims 6 to 8, characterized in that, Detect the fluorescence intensity at an excitation wavelength of 400 nm and an emission wavelength of 495 nm.

10. The application of the biosensor described in any one of claims 2 - 5 or the method described in any one of claims 6 - 8 in detecting neomycin residues in food.

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